PROJECT SUMMARY Graft versus host disease (GVHD) is the major complication associated with allogeneic hematopoietic stem cell transplantation (HSCT). Pathological damage to the skin, gastrointestinal tract, and liver are hallmarks of this disease; however, GVHD can also induce inflammation in the central nervous system (CNS) as well as cognitive and behavioral alterations in patients. We previously observed that host interleukin 6 (IL-6) production and the associated expansion of CNS resident macrophages (microglia) appear to have critical roles in the induction of neuroinflammation, but that blockade of IL-6 signaling does not completely mitigate disease severity. In preliminary studies, we have now identified endocannabinoid signaling through the type 2 cannabinoid receptor (CB2R) and the kynurenine metabolic pathway as novel IL-6- independent mechanisms by which inflammation is propagated in the brain. The overall goal of this proposal is to validate and characterize these two putative mechanistic pathways by which GVHD induces inflammation in the brain and ascertain how they modulate systemic manifestations of this disease. Our overall hypothesis is that inflammation during GVHD is attributable to CNS resident macrophages which induce inflammation and behavioral dysfunction through both the endocannabinoid signaling and kynurenine metabolic pathways. Studies in Specific Aim 1 will define the effect of CB2R signaling blockade on CNS and systemic manifestations of GVHD. We will employ pharmacological and genetic approaches that directly antagonize CB2R receptor signaling or inhibit the synthesis of 2-AG, the natural endocannabinoid ligand of the CB2R, to address this question. Experiments in Specific Aim 2 will determine whether expression of the CB2R on CNS resident macrophages is critical for mediating GVHD-induced inflammation. We will utilize novel CB2Rfl/fl mice which have flanking lox p sites which will allow for cell-specific deletion when bred with appropriate lineage-specific Cre animals. Studies in Specific Aim 3 will define the role of the kynurenine pathway in the pathophysiology of CNS and systemic inflammation that occur during GVHD. We will determine whether CNS resident macrophages are the dominant source of neurotoxic kynurenine metabolites, and define whether inhibition of this pathway using both genetic and pharmacological approaches prevents inflammation in the brain and periphery. The overall goal is to define relevant biochemical and immunological pathways that are responsible for GVHD-associated neuroinflammation in order to foster the development of clinically relevant strategies to mitigate this complication in allogeneic HSCT patients with blood cancers.